Cyclic prosaposin peptides and uses thereof

ABSTRACT

Provided herein are cyclic prosaposin peptides and compositions and uses thereof. Exemplary uses include use in the treatment of cancer or in the treatment of inflammatory diseases or disorders.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.16/854,792, filed Apr. 21, 2020, which is a Divisional of U.S. patentapplication Ser. No. 15/128,617, filed Sep. 23, 2016, which is the U.S.National Stage of International Patent Application No.PCT/US2015/022745, filed Mar. 26, 2015, which claims priority from U.S.Provisional Patent Application No. 61/970,853, filed Mar. 26, 2014. Thecontents of these applications are incorporated herein by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 23, 2016, isnamed 111827_0179_Sequence.txt and is 162,282 bytes in size.

GOVERNMENT SUPPORT

This invention was made with government support under R01CA135417awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF INVENTION

Cancer remains a major public health concern. For example, an estimated7.6 million deaths from cancer occurred in 2008. Many cancers are stilluntreatable or treatments are suboptimal, either being only partiallyeffective or having undesirable side-effects like toxicity.

Inflammatory diseases and disorders are also a major public healthconcern and also have similar treatment issues as cancer.

SUMMARY OF INVENTION

Aspects of the disclosure are based in part on the discovery that cyclicprosaposin peptides, such as cyclic peptides having the sequence DWLPK(SEQ ID NO: 1), are both more stable and more effective than linearprosaposin peptides for treatment of an animal model of cancer.Surprisingly, the cyclic DWLPK (SEQ ID NO: 1) peptide was found to bemore effective at stimulating thrombospondin-1 (Tsp-1) in vitro comparedto a cyclic peptide having a substitution of glycine at position 3(i.e., DWGPK, SEQ ID NO: 2). This data was unexpected because theopposite result was obtained with linear prosaposin peptides, in whichlinear DWGPK (SEQ ID NO: 2) was found to have better activity thanlinear DWLPK (SEQ ID NO: 1). Additionally, it was found that the cyclicDWLPK (SEQ ID NO: 1) peptide caused no toxicity to the liver or spleenof mice treated with the peptide.

Other aspects of the disclosure are based in part on the discovery thatcyclic DWLPK (SEQ ID NO: 1) is effective for treating a mouse model ofCrohn's disease.

Accordingly, aspects of the disclosure relate to cyclic prosaposinpeptides, e.g., DWLPK (SEQ ID NO: 1), as well as compositions andmethods utilizing such peptides.

Some aspects of the disclosure relate to a method for treating a subjecthaving cancer, the method comprises administering to a subject havingcancer an effective amount of a cyclic peptide to treat the cancer,wherein the amino acid sequence of the cyclic peptide is DWLPK (SEQ IDNO: 1). In some embodiments, the cancer is ovarian cancer or melanoma.

Other aspects relate to a composition for use in treating cancer, thecomposition comprising a cyclic peptide, wherein the amino acid sequenceof the cyclic peptide is DWLPK (SEQ ID NO: 1). In some embodiments, thecancer is ovarian cancer or melanoma.

Yet other aspects of the disclosure relate to a method for treating asubject having an inflammatory disease or disorder, the method comprisesadministering to a subject having an inflammatory disease or disorder aneffective amount of a cyclic peptide to treat the inflammatory diseaseor disorder, wherein the amino acid sequence of the cyclic peptide isDWLPK (SEQ ID NO: 1). In some embodiments, the inflammatory disease ordisorder is rheumatoid arthritis, age-related macular degeneration(AMD), Crohn's disease, psoriasis, or atherosclerosis. In someembodiments, the inflammatory disease or disorder is Crohn's disease.

Other aspects of the disclosure relate to a composition for use intreating an inflammatory disease or disorder, the composition comprisinga cyclic peptide, wherein the amino acid sequence of the cyclic peptideis DWLPK (SEQ ID NO: 1). In some embodiments, the inflammatory diseaseor disorder is rheumatoid arthritis, age-related macular degeneration(AMD), Crohn's disease, psoriasis, or atherosclerosis. In someembodiments, the inflammatory disease or disorder is Crohn's disease.

Other aspects of the disclosure relate to a method for stimulatingthrombospondin-1 (Tsp-1) expression, the method comprising:administering to a subject in need thereof an effective amount of acyclic peptide to stimulate Tsp-1 expression, wherein the amino acidsequence of the cyclic peptide is DWLPK (SEQ ID NO: 1).

Aspects of the disclosure relate to a cyclic Psap peptide. In someembodiments, the cyclic Psap peptide comprises the amino acid sequenceDWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicatesD-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower cased indicates D-amino acid). In some embodiments, the amino acid sequenceof the cyclic Psap peptide is DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3,lower case d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK(SEQ ID NO: 4, lower case d indicates D-amino acid). In someembodiments, the cyclic Psap peptide comprises the amino acid sequenceDWLPK (SEQ ID NO: 1). In some embodiments, the amino acid sequence ofthe cyclic Psap peptide is DWLPK (SEQ ID NO: 1).

Other aspects of the disclosure relate to compositions comprising acyclic Psap peptide as described herein. In some embodiments, thecomposition is a pharmaceutical composition. In some embodiments, thecomposition comprises a cyclic Psap peptide as described herein and apharmaceutically-acceptable carrier.

The details of one or more embodiments of the disclosure are set forthin the description below. Other features or advantages of the presentdisclosure will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is an exemplary schematic of cyclic DWLPK (SEQ ID NO: 1).

FIG. 2 is a photograph of a Western blot showing the level ofthrombospondin-1 (Tsp-1) activation in cells in response to differentcyclic or linear peptides (SEQ ID NOs: 1, 3, 2 and 4, respectively fromtop to bottom).

FIG. 3 is a graph showing the ability of cyclic DWLPK (SEQ ID NO: 1) orlinear dWIP SEQ ID NO: 5) to activate Tsp-1 after incubation in humanplasma.

FIG. 4 is a graph showing the tumor volume in mice treated with a cyclicpeptide or a linear peptide.

FIG. 5 is a graph showing the tumor volume in mice treated with a cyclicpeptide or a linear peptide.

FIG. 6 is a graph showing the total flux of luciferase-expressing tumorcells injected into mice after treatment with a cyclic peptide orcontrol.

FIG. 7 is a series of photographs showing that Tsp-1 is expressed inmacrophages that express F4/80.

FIG. 8 is a series of photographs showing the histology of liver andspleen samples in mice injected with control or cyclic DWLPK (SEQ ID NO:1).

FIG. 9 is a graph showing the MPO enzymatic activity (an index ofneutrophil infiltration into the colonic mucosa) in a Dextran SodiumSulfate (DSS)-induced mouse model of Crohn's disease treated withcontrol, untreated, or treated with cyclic DWLPK (SEQ ID NO: 1).

FIG. 10 is a graph showing the percent body weight in a mouse model ofCrohn's disease treated with control, untreated, or treated with cyclicDWLPK (SEQ ID NO: 1).

FIG. 11 is a series of photographs showing the histology of theintestine and macrophage localization in a mouse model of Crohn'sdisease treated with control, untreated, or treated with cyclic DWLPK(SEQ ID NO: 1).

FIGS. 12A-12E show stimulation of Tsp-1 and its effects on ovariancancer cell growth and survival. FIG. 12A is a western blot of Tsp-1 andβ-actin in WI-38 lung fibroblasts that were untreated (−) or treatedwith native DWLP (SEQ ID NO: 6), L-amino acid, prosaposin peptide (WT),dWIP (SEQ ID NO: 5, lower case d and 1 indicate D-amino acids)prosaposin peptide (d1.3) or DwLp (SEQ ID NO: 7, lower case w and pindicate D-amino acids) prosaposin peptide (d2,4): FIG. 128 is a westernblot of Tsp-1 and β-actin in mouse lung tissue harvested from micetreated with metastatic prostate cancer cell conditioned media alone (−)or in combination with DWLP (SEQ ID NO: 6) prosaposin peptide (WT) ordWIP (SEQ ID NO: 5, lower case d and l indicate D-amino acids)prosaposin peptide (d1,3 peptide) at doses of 10 and 30 mg/kg/day for 3days; FIG. 12C is a western blot of CD36 and β-actin in 8 patientderived ovarian cancer cell lines; FIG. 12D is a plot of cell number asmeasured by Wst-1 assay of a patient derived ovarian cancer cell linetreated with 1 μM recombinant human Tsp-1 (rhTsp-1) for 24, 48 and 72hours; and FIG. 12E is a FACS analysis of Annexin V and PI of a patientderived ovarian cancer cell line treated with 1 μM recombinant humanTsp-1 (rhTsp-1) for 48 hours.

FIGS. 13A-13E show the effects of a d-amino acid prosaposin peptide on aPDX model of metastatic ovarian cancer. FIG. 13A is a plot of relativeluciferase intensity of metastatic PDX ovarian tumors that were treatedwith saline (control), cisplatin 4 mg/kg/QOD (Cisplatin) and dWIP (SEQID NO: 5) prosaposin peptide (40 mg/kg QD); FIG. 13B shows luciferaseimaging of two control treated mice and two dWIP (SEQ ID NO: 5)prosaposin peptide treated mice at day 17 (treatment day 0) and day 48(treatment day 31), with each image having a luminescence scale to theright of the image that is shown in ×10⁸ units of radiance(p/sec/cm²/sr), with the minimum on the scale being 0.05e6 and themaximum on the scale being 1.00e8; FIG. 13C shows photographs of thelivers of mice bearing metastatic PDX ovarian tumors treated with saline(control) or dWIP (SEQ ID NO: 5) prosaposin peptide (Peptide); FIG. 13Dshows H&E staining of the liver of a mouse bearing metastatic PDXovarian tumors treated with saline (control) or dWIP (SEQ ID NO: 5)prosaposin peptide (Peptide). Right panels are 5× magnification and leftpanels are 20× magnification; and FIG. 13F, is a FACS analysis ofGR1⁺/Cd11b⁺ cells in the peritoneal fluid of mice control and dWIP (SEQID NO: 5) prosaposin peptide (Peptide) treated mice bearing metastaticPDX ovarian tumors after 48 days.

FIGS. 14A-14F show the effects of a cyclic prosaposin peptide on Tsp-1expression and a PDX model of metastatic ovarian cancer. FIG. 14A is awestern blot of Tsp-1 and β-actin in WI-38 lung fibroblasts that wereuntreated (−), treated with dWIP (SEQ ID NO: 5) prosaposin peptide(d1,3, “L”), or with cyclic DWLPK (SEQ ID NO: 1) prosaposin peptide(“C”): FIG. 14B shows an ELISA of Tsp-1 expression in WI-38 lungfibroblasts that were untreated (−), treated with dWIP (SEQ ID NO: 5)prosaposin peptide (d1,3), or with cyclic DWLPK (SEQ ID NO: 1)prosaposin peptide after up to 24 hours of incubation in human plasma at37° C.; FIG. 14C is a plot of relative luciferase intensity ofmetastatic PDX ovarian tumors that were treated with saline (control) orcyclic DWLPK (SEQ ID NO: 1) prosaposin peptide (10 mg/kg QD); FIG. 14Dis a plot of average metastatic lesion in saline (control) treated miceand cyclic DWLPK (SEQ ID NO: 1) prosaposin peptide (Peptide) treatedmice; FIG. 14E is an immunofluorescent staining of Gr1 and Tsp-1expression in metastatic lesions of control treated mice and cyclicDWLPK (SEQ ID NO: 1) prosaposin peptide (Peptide) treated mice; and FIG.14F shows the immunohistochemistry (leftmost panels) of Tsp-1 expressionand immunofluorescent staining of TUNEL and DAP1 of metastatic lesionsin control and cyclic DWLPK (SEQ ID NO: 1) prosaposin peptide (Peptide)treated mice.

FIGS. 15A-N show expression of CD36 and prosaposin in a TMA of humanovarian cancer patients. FIGS. 15A-15C show the expression of CD36 innormal human ovaries (Magnification: A=5×, B=10×, C=20×); FIGS. 15D-15Fshow the expression of CD36 in primary human ovarian tumors(Magnification: A=5×, B=10×, C=20×): FIGS. 15G-15I show the expressionof CD36 in human ovarian cancer metastases (Magnification: A=5×, B=10×,C=20×); FIGS. 15J-15L show the expression of CD36 in human ovariancancer lymph node metastases (Magnification: A=5×, B=10×, C=20×); FIG.15M is a plot of CD36 staining indices of normal human ovaries, primaryhuman ovarian tumors, human ovarian cancer metastases, and human ovariancancer lymph node metastases: and FIG. 15N is a plot of prosaposin(Psap) staining indices of normal human ovaries, primary human ovariantumors, human ovarian cancer metastases, and human ovarian cancer lymphnode metastases.

FIG. 16 shows that the psap peptide had no effect on body weight.

FIG. 17 shows pancreatic cancer cells expressing Tsp-1 receptors.

FIG. 18 shows that a cyclic prosaposin peptide DWLPK (SEQ ID NO: 1) hasapproximately 2-fold great activity than the linear peptide.

FIG. 19 shows that a cyclic prosaposin peptide DWLPK (SEQ ID NO: 1) hassignificant anti-tumor activity against pancreatic cancer.

FIG. 20 shows cyclic Psap peptide DWLPK (SEQ ID NO: 1) regressesmetastatic pancreatic cancer and inhibits metastasis.

DETAILED DESCRIPTION OF INVENTION

Prosaposin peptides (referred to herein as Psap peptides), which wereoriginally derived from fragments of Saposin A, were previously shown tobe effective for treating multiple types of cancers when used in linearform (see, e.g., PCT publications WO2009002931 WO/2011/084685 andWO/2013/096868, and U.S. patent application Ser. Nos. 12/640,788 and13/516,511, all of which are incorporated herein by reference in theirentirety).

As described herein, it has been discovered that cyclic Psap peptides,in particular cyclic DWLPK (SEQ ID NO: 1), are unexpectedly both morestable and more effective than linear Psap peptides. In addition, cyclicDWLPK (SEQ ID NO: 1) was found to have no toxic effect on the liver orspleen. Further, it was found that cyclic DWLPK (SEQ ID NO: 1) hadbetter activity than other cyclic versions of amino-acid-substitutedpeptides (e.g., DWGPK, SEQ ID NO: 2). This finding was surprisingbecause linear version of the substituted peptides were found to be moreactive than a linear version of DWLPK (SEQ ID NO: 1).

Also as described herein, it has been discovered that the cyclic Psappeptide DWLPK (SEQ ID NO: 1) was effective in a mouse model of Crohn'sdisease. Without wishing to be bound by theory, it is believed that thecyclic Psap peptide activates or enhances Tsp-1 expression and that theTsp-1 expression reduces inflammation associated with Crohn's disease.Tsp-1 has been shown previously to be secreted in response toinflammation, promoting the resolution of the inflammatory process andfacilitating phagocytosis of damaged cells (see, e.g., Lopez-Dee et al.Thrombospondin-1: Multiple Paths to Inflammation. (2011) Mediators ofInflammation; Vol. 2011; Article ID 296069). Thus, as a therapeuticeffect of the cyclic Psap peptide was seen in a model of Crohn'sdisease, which is an exemplary inflammatory disease, it is believed thatthe cyclic Psap peptide will also be effective for treating otherinflammatory diseases, such as rheumatoid arthritis and psoriasis, aswell as other diseases in which inflammation is involved, e.g.,inflammatory disorders such as atherosclerosis, and age-related maculardegeneration (AMD).

Accordingly, aspects of the disclosure relate to cyclic Psap peptides,as well as their use in compositions and methods for treating diseases,such as cancer and inflammatory disorders or diseases as well as otherdiseases in which inflammation is involved.

Cyclic Psap Peptides

Prosaposin (Psap) is the Saposin precursor protein made up ofapproximately 524-527 amino acids which includes a 16 amino acids signalpeptide. The full-length precursor polypeptide undergoesco-translational glycosylation and modification in the endoplasmicreticulum and Golgi system to yield a 70-72 kDa precursor protein. Aftertransport to the lysosome, cathepsin D participates in its proteolyticprocessing to yield intermediate molecular forms of 35 to 53 kDa andthen to a 13-kDa glycoprotein and finally to the mature 8-11 kDapartially glycosylated forms of individual Saposin molecules (O'Brien J.S., and Kishimoto Y, The FASEB J., 5: 301-8, 1991; Kishimoto Y. et al.,J. Lipid Res. 33:1255-67, 1992). Prosaposin is processed into 4 cleavageproducts: Saposins A, B, C, and D. The amino acid sequences of Psappreproprotein isoforms A, B, and C and the amino acid sequence ofcleavage product Saposin A are below:

Psap Preproprotein Isoform A (SEQ ID NO: 9)MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN Psap Preproprotein Isoform B(SEQ ID NO: 10) MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTYKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHLAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMQDQQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN Psap Preproprotein Isoform C(SEQ ID NO: 11) MYALFLLASLLGAALAGPVLGLKECTRGSAVWCQNVKTASDCGAVKHCLQTVWNKPTVKSLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCESLQKHILAELNHQKQLESNKIPELDMTEVVAPFMANIPLLLYPQDGPRSKPQPKDNGDVCQDCIQMVTDIQTAVRTNSTFVQALVEHVKEECDRLGPGMADICKNYISQYSEIAIQMMMHMDQQPKEICALVGFCDEVKEMPMQTLVPAKVASKNVIPALELVEPIKKHEVPAKSDVYCEVCEFLVKEVTKLIDNNKTEKEILDAFDKMCSKLPKSLSEECQEVVDTYGSSILSILLEEVSPELVCSMLHLCSGTRLPALTVHVTQPKDGGFCEVCKKLVGYLDRNLEKNSTKQEILAALEKGCSFLPDPYQKQCDQFVAEYEPVLIEILVEVMDPSFVCLKIGACPSAHKPLLGTEKCIWGPSYWCQNTETAAQCNAVEHCKRHVWN Saposin A (SEQ ID NO: 12)SLPCDICKDVVTAAGDMLKDNATEEEILVYLEKTCDWLPKPNMSASCKEIVDSYLPVILDIIKGEMSRPGEVCSALNLCES

Aspects of the disclosure relate to a cyclic Psap peptide, compositionscomprising a cyclic Psap peptide, and uses thereof. In some embodiments,the cyclic Psap peptide comprises the amino acid sequence DWLPK (SEQ IDNO: 1), dWLPK (SEQ ID NO: 3, lower cased indicates D-amino acid), DWGPK(SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower case d indicates D-aminoacid). In some embodiments, the amino acid sequence of the cyclic Psappeptide is DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case dindicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4,lower case d indicates D-amino acid). In some embodiments, the cyclicPsap peptide comprises the amino acid sequence DWLPK (SEQ ID NO: 1). Insome embodiments, the amino acid sequence of the cyclic Psap peptide isDWLPK (SEQ ID NO: 1).

In some embodiments, the cyclic peptide is less than 10, less than 9,less than 8, less than 7, less than 6, or 5 amino acids in length. Insome embodiments, the cyclic peptide is between 5-10, 5-9, 5-8, 5-7 or5-6 amino acids in length. In some embodiments, the cyclic peptide is 5amino acids in length.

Peptides described herein can be chemically synthesized and isolated bybiochemical methods that are well known in the art such as solid phasepeptide synthesis using t-Boc (tert-butyloxycarbonyl) or FMOC(9-flourenylmethloxycarbonyl) protection group described in “Peptidesynthesis and applications” in Methods in molecular biology Vol. 298,Ed. by John Howl and “Chemistry of Peptide Synthesis” by N. LeoBenoiton, 2005, CRC Press, (ISBN-13: 978-15711/4544) and “ChemicalApproaches to the Synthesis of Peptides and Proteins” by P.Lloyd-Williams, et al. 1997, CRC-Press, (ISBN-13: 978-0849391422). Solidphase peptide synthesis, developed by R. B. Merrifield, 1963, J. Am.Chem. Soc. 85 (14): 2149-2154, was a major breakthrough allowing for thechemical synthesis of peptides and small proteins.

Cyclic Psap peptides can be synthesized using any method known in theart. Exemplary methods of synthesis include, but are not limited to,recombinant synthesis, liquid-phase synthesis, Solid-phase synthesis, orchemical ligation (see, e.g., Molecular Cloning: A Laboratory Manual, J.Sambrook, et al., eds., Third Edition, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001; Current Protocols in MolecularBiology. F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York;Schnolzer, M. A., P.; Jones, A.; Alewood, D.; Kent, S. B. H. (2007). “InSitu Neutralization in Boc-chemistry Solid Phase Peptide Synthesis”.Int. J. Peptide Res. Therap. 13 (1-2): 31-44; Albericio, F. (2000).Solid-Phase Synthesis: A Practical Guide (1 ed.). Boca Raton: CRC Press.p. 848; and Nilsson B L, Soellner M B, Raines R T (2005). “ChemicalSynthesis of Proteins”. Annu. Rev. Biophys. Biomol. Struct. 34: 91-118;and U.S. Pat. Nos. 4,749,742, 4,794,150, 5,552,471, 5,637,719,6,001,966, 7,038,103, 7,094,943, 7,176,282, and 7,645,858, the entiretyof which are incorporated herein by reference). Nap peptides and methodsof making Psap peptides are also known in the art (see, e.g., PCTpublications WO2009002931, WO/2011/084685 and WO/2013/096868, all ofwhich are incorporated herein by reference in their entirety). Cyclicpeptides are polypeptide chains that are linked together with a peptidebond or other covalent bond, forming a circular structure. Methods ofdesign and synthesis of cyclic peptides are well known in the art (see,e.g. as described in U.S. Pat. Nos. 5,596,078; 5,990,273; 7,589,170 andU.S. Patent Application No. 20080287649) and commercially available(see, e.g., cyclic peptide synthesis services offered by SelleckChemicals, Abbiotec, Agent, AnaSpec Global Peptide Services, LLC.,INVITROGENT™ and rPeptide, LLC or GenScript). Cyclic peptides may becircularized. e.g., head-to-tail (C-terminus to N-terminus),head-to-side chain, side-chain-to-tail, or side-chain-to-side-chain(see, e.g., White et al. Contemporary strategies for peptidemacrocyclization. Nature Chemistry (2011); 3:509-524). A cyclizationreagent, such as PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate). BOP(benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphoniumhexa-fluorophosphate), DIPEA (N,N-diisopropylethylamine), DMTMM BF4(4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholiniumtetrafluoroborate) or FDPP (pentafluorophenyl diphenylphosphinate) orcombinations thereof, may be used during peptide synthesis or afterpeptide synthesis to induce circulization. An exemplary method involvesuse of a photolabile auxiliary. e.g., HnB (2-hydroxy-6-nitrobenzyl), ina ring-contraction strategy to cyclize a pentapeptide. After theintroduction of this auxiliary onto the N-terminus of the peptide, thepeptide is contacted with BOP and DIPEA, resulting an intramolecularO-to-N acyl transfer, which in turn results in the cyclic pentapeptide.The photolabile auxiliary can then be removed.

In some embodiments, the Psap peptide may be modified, for example,through oligomerization or polymerization (e.g., dimers, trimer,multimers, etc.), modifications of amino acid residues or peptidebackbone, cross-linking, conjugation, pegylation, glycosylation,acetylation, phosphorylation, fusion to additional heterologous aminoacid sequences (for example, an antibody or antibody Fe domain, serumtransferrin or portions thereof, albumin, or transthyretin), or othermodifications that substantially alter the stability, solubility, orother properties of the peptide while substantially retaining orenhancing therapeutic activity. Conjugation may be, e.g., to a polymer.Suitable polymers include, for example, polyethylene glycol (PEG),polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylethermaleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextranderivatives including dextran sulfate, polypropylene glycol,polyoxyethylated polyol, heparin, heparin fragments, polysaccharides,cellulose and cellulose derivatives, including methylcellulose andcarboxymethyl cellulose, starch and starch derivatives, polyalkyleneglycol and derivatives thereof, copolymers of polyalkylene glycols andderivatives thereof, polyvinyl ethyl ethers, andα,β-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixturesthereof. Conjugation may be through a linker, e.g., a peptide orchemical linker. Methods of modifying peptides are well known in the art(see, e.g., U.S. Pat. Nos. 5,180,816, 5,596,078, 5,990,273, 5,766,897,5,856,456, 6,423,685, 6,884,780, 7,610,156, 7,256,258, 7,589,170 and7,022,673, and PCT publication WO 2010/014616, the contents of which areincorporated herein by reference).

In some embodiments, the cyclic Psap peptide is functionally modified toenhance stability. In some embodiments, chemical modifications to thePsap peptide include, but are not limited to the inclusion of, alkyl,alkoxy, hydroxyalkyl, alkoxyalkyl, alkoxycarbonyl, alkenyl, alkynyl,cycloalkyl, amino, alkylamino, aminoalkyl, dialkylamino, aminodialkyl,halogen, heteroatom, carbocycle, carbocyclyl, carbocyclo, carbocyclic,aryl, aralkyl, aralkoxy, aryloxyalkyl, heterocycle, heterocyclyl,heterocyclic, heteroaryl, and/or aliphatic groups.

In some embodiments, the cyclic peptide is fused/conjugated to at leastone therapeutic molecule.

In some embodiments, amino acid substitution variants of a cyclic Psappeptide resulting from substitution of one or more D-amino acids for thelike L-amino acid are contemplated herein. In some embodiments, oneD-amino acid substitution is present. In some embodiments, 2 or moreD-amino acid substitutions are present. In some embodiments, 3, 4, or 5D-amino acid substitutions are present. In some embodiments, the D-aminoacid substitutions are evenly spaced, e.g., every other amino acid. Insome embodiments, the D-amino acid substitution is for Aspartic Acid(D). The L and D convention for amino acid configuration refers not tothe optical activity of the amino acid itself, but rather to the opticalactivity of the isomer of glyceraldehyde from which that amino acid can,in theory, be synthesized (D-glyceraldehyde is dextrorotary;L-glyceraldehyde is levorotary). Exemplary D amino acid substitutionvariants include cyclic dWLPK (SEQ ID NO: 3, lower case d indicatesD-amino acid) and cyclic dWGPK (SEQ ID NO: 4, lower case d indicatesD-amino acid).

The cyclic peptide described herein can further be modified orderivatized. The modified or derivatized polypeptides will typicallysubstantially retain the activity of the base polypeptide(pre-modified/derivatized). Examples of modifications and derivativesare pegylation, glycosylation, acetylation, amidation, andphosphorylation. Methods of acetylation (e.g., N-terminal acetylation)and amidation (e.g., C-terminal amidation) are well known to those ofskill in the art. Modifications, derivatives and methods of derivatizingpolypeptides are described in Published International Application WO2010/014616, the contents of which are incorporated herein by reference.

In some embodiments, the cyclic peptide described herein can beconjugated or otherwise covalently attached to other molecules (e.g.,using a chemical linker). One such form of attachment is through anon-amide linkage (e.g., a disulfide bond). In some embodiments, thecyclic peptide is linked to a polymer that enhances the serum half-life.In some embodiments, the cyclic peptide is covalently attached (e.g.,via a linker molecule) to an antibody or a domain thereof suitable forenhancing the half-life of the molecule (e.g., one or more constantdomains in an Fc domain). In some embodiments, the polypeptide is linkedto an Fc domain (e.g., IgG, IgA, IgM. IgD, or IgE).

In some embodiments, the cyclic peptide described herein is linked to anon-amino acid polymer. Polymers such as polyethylene glycol can be usedfor the purpose of enhancing the serum half-life. Suitable polymersinclude, for example, polyethylene glycol (PEG), polyvinyl pyrrolidone,polyvinyl alcohol, polyamine acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polyoxyethylatedpolyol, heparin, heparin fragments, polysaccharides, cellulose andcellulose derivatives, including methylcellulose and carboxymethylcellulose, starch and starch derivatives, polyalkylene glycol andderivatives thereof, copolymers of polyalkylene glycols and derivativesthereof, polyvinyl ethyl ethers, andα,β-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixturesthereof. Such a polymer may or may not have its own biological activity.The polymers can be covalently or non-covalently conjugated to thecyclic peptide. Methods of conjugation for increasing serum half-lifeare known in the art, for example, in U.S. Pat. Nos. 5,180,816,6,423,685, 6,884,780, and 7,022,673, which are hereby incorporated byreference in their entirety.

In some embodiments, the cyclic peptide is conjugated to a therapeuticmolecule. In some embodiments, the therapeutic molecule is ananti-angiogenic therapeutic molecule, an anti-VEGF agent, achemotherapeutic agent, or an anti-inflammatory agent.

The various versions of cyclic peptides described herein, e.g.,modified, conjugated, or derivatized cyclic peptides, encompassed by thepresent disclosure are expected to retain a significant amount of thebiological activity exhibited by the cyclic peptide (e.g., as reportedin the Examples section herein). In some embodiments, about 100% of theactivity is retained in a given assay. In some embodiments, about 90%,80%, 70%, 60% or 50% of the activity is retained. One such activity isthe ability to stimulate expression of Tsp-1. Stimulation of expressionis a significant, reproducible amount of increased expression thatoccurs from contact of the cyclic peptide described herein with anappropriate target cell, as compared to an identical or sufficientlysimilar target cell (control target cell) that has not been contactedwith the cyclic peptide. The methods for determining Tsp-1 expressioninduction activity are described herein and are also well known to oneskilled in the art.

The amino acid abbreviations are shown below.

Alanine, Ala, A

Isoleucine, He, I

Leucine, Leu, L

Valine, Val, V

Phenylalanine, Phe, F

Tryptophan, Trp, W

Tyrosine, Tyr, Y

Asparagine, Asn, N

Cysteine, Cys, C

Glutamine, Gin, Q

Methionine, Met, M

Serine, Ser, S

Threonine, Thr, T

Aspartic acid, Asp, D

Glutamic acid, Glu, E

Arginine, Arg, R

Histidine, His, H

Lysine, Lys, K

Glycine, Gly, G

Proline, Pro, P

Other Cyclic Peptide Modifications

It is to be understood that modified versions of the cyclic peptidesdescribed herein are encompassed in the present disclosure. Modificationto a cyclic peptide described herein can be performed as described inU.S. published application 20080090760 and/or U.S. published application20060286636, each of which is incorporated herein by reference in itsentirety. The following provides a non-limiting discussion of variousother peptide modifications encompassed within the scope of thedisclosure.

Encompassed by the present disclosure are chemical derivatives of acyclic peptide described herein, so long as it substantially retains theactivities of the non-derivatized cyclic peptide. A “chemicalderivative” is a subset of peptide derivatives as described herein andrefers to a subject cyclic peptide having one or more residueschemically derivatized by reaction of a functional side group. Inaddition to side group derivatizations, a chemical derivative can haveone or more backbone modifications including alpha-amino substitutionssuch as N-methyl, N-ethyl. N-propyl and the like, and alpha-carbonylsubstitutions such as thioester, thioamide, guanidino and the like. Suchderivatized molecules include for example, those molecules in which freeamino groups have been derivatized to form amine hydrochlorides,p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonylgroups, chloroacetyl groups or formyl groups. Free carboxyl groups maybe derivatized to form salts, methyl and ethyl esters or other types ofesters or hydrazides. Free hydroxyl groups may be derivatized to formO-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine maybe derivatized to form N-im-benzylhistidine. Also included as chemicalderivatives are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids.Also included as chemical derivatives are those peptides which containone or more non-limiting, non-natural amino acids, examples includethose available for peptide synthesis from commercial suppliers (e.g.Bachem Catalog, 2004 pp. 1-276). For examples: 4-hydroxyproline may besubstituted for proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; ornithine may be substituted for lysine;β-alanine may be substituted for alanine; norleucine may be substitutedfor leucine; phenylglycine may be substituted for phenylalanine, andL-1,2,3,4-tetrahydronorharman-3-carboxylic acid orH-β-(3-Benzothienyl)-Ala-OH may be substituted for tryptophan.

In certain embodiments, chemical modifications to the peptide include,but are not limited to the inclusion of, alkyl, alkoxy, hydroxyalkyl,alkoxyalkyl, alkoxycarbonyl, alkenyl, alkynyl, cycloalkyl, amino,alkylamino, aminoalkyl, dialkylamino, aminodialkyl, halogen, heteroatom,carbocycle, carbocyclyl, carbocyclo, carbocyclic, aryl, aralkyl,aralkoxy, aryloxyalkyl, heterocycle, heterocyclyl, heterocyclic,heteroaryl, and/or aliphatic groups.

The terms “alkyl”, “alkoxy”, “hydroxyalkyl”, “alkoxyalkyl”, and“alkoxycarbonyl”, used alone or as part of a larger moiety includes bothstraight and branched chains containing one to twelve carbon atoms. Theterms “alkenyl” and “alkynyl” used alone or as part of a larger moietyshall include both straight and branched chains containing two to twelvecarbon atoms. The term “cycloalkyl” used alone or as part of a largermoiety shall include cyclic C3-C12 hydrocarbons which are completelysaturated or which contain one or more units of unsaturation, but whichare not aromatic. Lower alkyl refers to an alkyl group containing 1-6carbons.

The term “amino” refers to an NH2 group. The term “alkylamino” or“aminoalkyl” refers to an amino group wherein one of the hydrogen atomsis replaced by an alkyl group. The term “dialkylamino” or “aminodialkyl”refers to an amino group wherein the hydrogen atoms are replaced byalkyl groups, wherein the alkyl group may be the same or different. Theterm “halogen” means F, Cl, Br, or I. The term “heteroatom” meansnitrogen, oxygen, or sulfur with a carbon ring structure and includesany oxidized form of nitrogen and sulfur, and the quaternized form ofany basic nitrogen. Also the term “nitrogen” includes a substitutablenitrogen of a heterocyclic ring. As an example, in a saturated orpartially unsaturated ring having 0-3 heteroatoms selected from oxygen,sulfur or nitrogen, the nitrogen may be N (as in3,4-dihydro-2H-pyrrolyl). NH (as in pyrrolidinyl) or NR+ (as inN-substituted pyrrolidinyl). The terms “carbocycle”, “carbocyclyl”,“carbocyclo”, or “carbocyclic” as used herein means an aliphatic ringsystem having three to fourteen members. The terms “carbocycle”,“carbocyclyl”, “carbocyclo”, or “carbocyclic” whether saturated orpartially unsaturated, also refers to rings that are optionallysubstituted. The terms “carbocycle”, “carbocyclyl”, “carbocyclo”, or“carbocyclic” also include aliphatic rings that are fused to one or morearomatic or nonaromatic rings, such as in a decahydronaphthyl ortetrahydronaphthyl, where the radical or point of attachment is on thealiphatic ring.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to aromatic ring groupshaving six to fourteen members, such as phenyl, benzyl, phenethyl,1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl. The term “aryl”also refers to rings that are optionally substituted. The term “aryl”may be used interchangeably with the term “aryl ring”. “Aryl” alsoincludes fused polycyclic aromatic ring systems in which an aromaticring is fused to one or more rings. Examples include 1-naphthyl,2-naphthyl, 1-anthracyl and 2-anthracyl. Also included within the scopeof the term “aryl”, as it is used herein, is a group in which anaromatic ring is fused to one or more non-aromatic rings, such as in anindanyl, phenanthridinyl, or tetrahydronaphthyl, where the radical orpoint of attachment is on the aromatic ring.

The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used hereinincludes non-aromatic ring systems having four to fourteen members,preferably five to ten, in which one or more ring carbons, preferablyone to four, are each replaced by a heteroatom. Examples of heterocyclicrings include 3-1H-benzimidazol-2-one,(1-substituted)-2-oxo-benzimidazol-3-yl, 2-tetrahydro-furanyl,3-tetrahydrofuranyl, 2-tetrahydropyranyl, 3-tetrahydropyranyl,4-tetra-hydropyranyl, [1,3]-dioxalanyl, [1,3]-dithiolanyl,[1,3]-dioxanyl, 2-tetra-hydro-thiophenyl, 3-tetrahydrothiophenyl,2-morpholinyl, 3-morpholinyl, 4-morpholinyl, 2-thiomorpholinyl,3-thiomorpholinyl, 4-thiomorpholinyl, 1-pyrrolidinyl, 2 pyrrolidinyl,3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 4-thiazolidinyl,diazolonyl, N-substituted diazolonyl, 1-phthalimidinyl, benzoxanyl,benzopyrrolidinyl, benzopiperidinyl, benzoxolanyl, benzothiolanyl, andbenzothianyl. Also included within the scope of the term “heterocyclyl”or “heterocyclic”, as it is used herein, is a group in which anon-aromatic heteroatom-containing ring is fused to one or more aromaticor non-aromatic rings, such as in an indolinyl, chromanyl,phenanthridinyl, or tetrahydroquinolinyl, where the radical or point ofattachment is on the non-aromatic heteroatom-containing ring. The term“heterocycle”, “heterocyclyl”, or “heterocyclic” whether saturated orpartially unsaturated, also refers to rings that are optionallysubstituted.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to heteroaromatic ringgroups having five to fourteen members. Examples of heteroaryl ringsinclude 2-furanyl, 3-furanyl, 3-furazanyl, N-imidazolyl, 2-imidazolyl,4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,2-oxadiazolyl. 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl. 5-oxazolyl,1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 1-pyrazolyl, 2-pyrazolyl,3-pyrazolyl, 2-pyridyl, 3-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-pyrimidyl, 3-pyridazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl, 3-thienyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, indazolyl, isoindolyl, acridinyl, andbenzoisoxazolyl. Also included within the scope of the term“heteroaryl”, as it is used herein, is a group in which a heteroatomicring is fused to one or more aromatic or nonaromatic rings where theradical or point of attachment is on the heteroaromatic ring. Examplesinclude tetrahydroquinolinyl, tetrahydroisoquino-linyl, andpyrido[3,4-d]pyrimidinyl. The term “heteroaryl” also refers to ringsthat are optionally substituted. The term “heteroaryl” may be usedinterchangeably with the term “heteroaryl ring” or the term“heteroaromatic”.

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) orheteroaryl (including heteroaralkyl and heteroarylalkoxy and the like)group may contain one or more substituents. Examples of suitablesubstituents on any unsaturated carbon atom of an aryl, heteroaryl,aralkyl, or heteroaralkyl group include a halogen, —R0, —OR0, —SR0,1,2-methylene-dioxy, 1,2-ethylenedioxy, protected OH (such as acyloxy),phenyl (Ph), substituted Ph, —O(Ph), substituted —O(Ph), —CH2(Ph),substituted —CH2(Ph), CH2CH2(Ph), substituted —CH2CH2(Ph), —NO2, —CN,—N(R0)2, —NR0C(O)R0, NR0C(O)N(R0)2, NR0CO2R0, —NR0NR0C(O)R0,—NR0NR0C(O)N(R0)2, —NR0NR0C2R0, C(O)C(O)R0, C(O)CH2C(O)R0, —CO2R0,—C(O)R0, —C(O)N(R0)2, —OC(O)N(R0)2, S(O)2R0, —SO2N(R0)2, —S(O)R0,—NR0SO2N(R0)2, —NR0SO2R0, —C(═S)N(R0)2, C(═NH)N(R0)2, (CH2)yNHC(O)R0,and —(CH2)yNHC(O)CH(V—R0)(R0); wherein each R0 is independently selectedfrom hydrogen, a substituted or unsubstituted aliphatic group, anunsubstituted heteroaryl or heterocyclic ring, phenyl (Ph), substitutedPh, O(Ph), substituted —O(Ph), —CH2 (Ph), or substituted —CH2(Ph); y is0-6; and V is a linker group. Examples of substituents on the aliphaticgroup or the phenyl ring of R0 include amino, alkylamino, dialkylamino,aminocarbonyl, halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano,carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, andhaloalkyl.

An aliphatic group or a non-aromatic heterocyclic ring or a fused arylor heteroaryl ring may contain one or more substituents. Examples ofsuitable substituents on any saturated carbon of an aliphatic group orof a non-aromatic heterocyclic ring or a fused aryl or heteroaryl ringinclude those listed above for the unsaturated carbon of an aryl orheteroaryl group and the following: ═O, ═S, ═NNHR*, ═NN(R*)2, ═N—,═NNHC(O)R*, ═NNHCO2(alkyl), ═NNHSO2 (alkyl), or ═NR*, where each R* isindependently selected from hydrogen, an unsubstituted aliphatic group,or a substituted aliphatic group. Examples of substituents on thealiphatic group include amino, alkylamino, dialkylamino, aminocarbonyl,halogen, alkyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylaminocarbonyloxy, dialkylaminocarbonyloxy, alkoxy, nitro, cyano,carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy, haloalkoxy, andhaloalkyl.

Suitable substituents on the nitrogen of a non-aromatic heterocyclicring include R+, —N(R+)2, —C(O)R+, —CO2R+, —C(O)C(O)R+, —C(O)CH2C(O)R+,—SO2R+, —SO2N(R+)2, C(═S)N(R+)2, —C(═NH)—N(R+)2, and —NR+SO2R+; whereineach R+ is independently selected from hydrogen, an aliphatic group, asubstituted aliphatic group, phenyl (Ph), substituted Ph, —O(Ph),substituted —O(Ph). —CH2(Ph), substituted —CH2(Ph), or an unsubstitutedheteroaryl or heterocyclic ring. Examples of substituents on thealiphatic group or the phenyl ring include amino, alkylamino,dialkylamino, aminocarbonyl, halogen, alkyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylaminocarbonyloxy, dialkylaminocarbonyloxy,alkoxy, nitro, cyano, carboxy, alkoxycarbonyl, alkylcarbonyl, hydroxy,haloalkoxy, and haloalkyl.

In certain embodiments, the peptide monomers described herein aredimerized or multimerized by covalent attachment to at least one linkermoiety. The linker moiety is preferably, although not necessarily, aC1-12 linking moiety optionally terminated with one or two —NH— linkagesand optionally substituted at one or more available carbon atoms with alower alkyl substituent. Preferably the linker comprises —NH—R—NH—wherein R is a lower (C1-6) alkylene substituted with a functionalgroup, such as a carboxyl group or an amino group, that enables bindingto another molecular moiety (e.g., as may be present on the surface of asolid support during peptide synthesis or to a pharmacokinetic-modifyingagent such as PEG). In certain embodiments the linker is a lysineresidue. In certain other embodiments, the linker bridges the C-terminiof two peptide monomers, by simultaneous attachment to the C-terminalamino acid of each monomer. In other embodiments, the linker bridges thepeptides by attaching to the side chains of amino acids not at theC-termini. When the linker attaches to a side chain of an amino acid notat the C-termini of the peptides, the side chain preferably contains anamine, such as those found in lysine, and the linker contains two ormore carboxy groups capable of forming an amide bond with the peptides.

The peptide monomers of the disclosure may be oligomerized using thebiotin/streptavidin system. Oligomerization can enhance one or moreactivities of peptides as described herein. Biotinylated analogs ofpeptide monomers may be synthesized by standard techniques known tothose skilled in the art. For example, the peptide monomers may beC-terminally biotinylated. These biotinylated monomers are thenoligomerized by incubation with streptavidin (e.g., at a 4:1 molar ratioat room temperature in phosphate buffered saline (PBS) or HEPES-bufferedRPM1 medium (Invitrogen) for 1 hour). In a variation of this process,biotinylated peptide monomers may be oligomerized by incubation with anyone of a number of commercially available anti-biotin antibodies [e.g.,goat anti-biotin IgG from Kirkegaard & Perry Laboratories, Inc.(Washington. D.C.)].

In some aspects, the cyclic peptides described herein can be linkedphysically in tandem to form a polymer. The cyclic peptides making upsuch a polymer can be spaced apart from each other by a peptide linker.Examples of useful linker peptides include, but are not limited to,glycine polymers ((G)n) including glycine-serine and glycine-alaninepolymers (e.g., a (Gly4Ser)n repeat where n=1-8 (SEQ ID NO: 8),preferably, n=3, 4, 5, or 6). The cyclic peptides described herein canalso be joined by chemical bond linkages, such as linkages by disulfidebonds or by chemical bridges. Molecular biology techniques that are wellknown to those skilled in the art can be used to create a polymer of thecyclic peptides. Peptide sequences of the present disclosure can also belinked together using non-peptide cross-linkers (Pierce 2003-2004Applications Handbook and Catalog. Chapter 6) or other scaffolds such asHPMA, polydextran, polysaccharides, ethylene-glycol,poly-ethylene-glycol, glycerol, sugars, and sugar alcohols (e.g.sorbitol, mannitol).

In some embodiments, polyethylene glycol (PEG) may serve as a linker. Inyet another embodiment, a linker moiety may comprise a moleculecontaining two carboxylic acids and optionally substituted at one ormore available atoms with an additional functional group such as anamine capable of being bound to one or more PEG molecules. Such amolecule can be depicted as: —CO—(CH2)n-uX—(CH2)m-CO— where n is aninteger between zero and 10, m is an integer between one and 10, X isselected from O, S, N(C112)pNR1, NCO(CH2)pNR1, and CHNR1, R1 is selectedfrom H, Boc (tert-butyloxycarbonyl), Cbz, and p is an integer between 1and 10. In certain embodiments, one amino group of each of the peptidesforms an amide bond with the linker. Optionally, the linker contains oneor more reactive amines capable of being derivatized with a suitablyactivated pharmacokinetic (PK) modifying agent such as a fatty acid, ahoming peptide, a transport agent, a cell-penetrating agent, anorgan-targeting agent, or a chelating agent.

The cyclic peptide described herein may further comprise one or morewater soluble polymer moieties. In some embodiments, these polymers arecovalently attached to the cyclic peptide of the disclosure. In someembodiments, for therapeutic use of the end product preparation, thepolymer is pharmaceutically acceptable. One skilled in the art will beable to select the desired polymer based on such considerations aswhether the polymer-peptide conjugate will be used therapeutically, andif so, the desired dosage, circulation time, resistance to proteolysis,and other considerations. The water soluble polymer may be, for example,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), poly(n-vinyl-pyrrolidone)polyethylene glycol, propropyleneglycol homopolymers, polypropylene oxide/ethylene oxide copolymers, andpolyoxyethylated polyols. A preferred water soluble polymer is PEG.

The polymer may be of any molecular weight, and may be branched orunbranched. A preferred PEG for use in the present disclosure is linear,unbranched PEG having a molecular weight of from about 5 kilodaltons(kDa) to about 60 kDa (the term “about” indicating that in preparationsof PEG, some molecules will weigh more, and some less, than the statedmolecular weight). More preferably, the PEG has a molecular weight offrom about 10 kDa to about 40 kDa, and even more preferably, the PEG hasa molecular weight from 20 to 30 kDa. Other sizes may be used, dependingon the desired therapeutic profile (e.g., duration of sustained releasedesired; effects, if any, on biological activity; ease in handling;degree or lack of antigenicity; and other effects of PEG on atherapeutic peptide known to one skilled in the art).

The number of polymer molecules attached may vary; for example, one,two, three, or more water-soluble polymers may be attached to a peptideof the disclosure. The multiple attached polymers may be the same ordifferent chemical moieties (e.g., PEGs of different molecular weight).

Methods for stabilizing peptides known in the art may be used with themethods and compositions described herein. For example, using D-aminoacids, using reduced amide bonds for the peptide backbone, and usingnon-peptide bonds to link the side chains, including, but not limitedto, pyrrolinone and sugar mimetics can each provide stabilization. Thedesign and synthesis of sugar scaffold peptide mimetics are described byHirschrnann et al. (J. Med. Chem., 1996, 36, 2441-2448, which isincorporated herein by reference in its entirety). Further,pyrrolinone-based peptide mimetics present the peptide pharmacophore ona stable background that has improved bioavailability characteristics(see, for example, Smith et al., J. Am. Chem. Soc. 2000, 122,11037-11038), which is incorporated herein by reference in its entirety.

Encompassed herein are conjugates of the cyclic described herein. Thesepeptides can be conjugated to other polymers in addition to polyethyleneglycol (PEG). The polymer may or may not have its own biologicalactivity. Further examples of polymer conjugation include but are notlimited to polymers such as polyvinyl pyrrolidone, polyvinyl alcohol,polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polyoxyethylatedpolyol, heparin, heparin fragments, polysaccharides, cellulose andcellulose derivatives, including methylcellulose and carboxymethylcellulose, starch and starch derivatives, polyalkylene glycol andderivatives thereof, copolymers of polyalkylene glycols and derivativesthereof, polyvinyl ethyl ethers, andα,β-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixturesthereof. Conjugation to a polymer can improve serum half-life, amongother effects. A variety of chelating agents can be used to conjugatethe peptides described herein. These chelating agents include but arenot limited to ethylenediaminetetraacetic acid (EDTA),diethylenetriaminopentaacetic acid (DTPA),ethyleneglycol-0,0′-bis(2-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA),N,N′-bis(hydroxybenzyl)ethylenediamine-N,N′-diacetic acid (HBED),triethylenetetraminehexaacetic acid (TTHA),1,4,7,10-tetra-azacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA),1,4,7,10-tetraazacyclotridecane-1,4,7,10-tetraacetic acid (TITRA),1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N′″-tetraacetic acid (TETA),and 1,4,8,11-tetraazacyclotetradecane (TETRA). Methods of conjugationare well known in the art, for example, P. E. Thorpe, et. al, 1978,Nature 271, 752-755; Harokopakis E., et. al., 1995, Journal ofImmunological Methods, 185:31-42; S. F. Atkinson, et. al., 2001, J.Biol. Chem., 276:27930-27935; and U.S. Pat. Nos. 5,601,825, 5,180,816,6,423,685, 6,706,252, 6,884,780, and 7,022,673, which are herebyincorporated by reference in their entirety.

One can replace the naturally occurring side chains of the geneticallyencoded amino acids (or the stereoisomeric D amino acids) with otherside chains, for instance with groups such as alkyl, lower (C1-6) alkyl,cyclic 4-, 5-, 6-, to 7-membered alkyl, amide, amide lower alkyl amidedi(lower alkyl), lower alkoxy, hydroxy, carboxy and the lower esterderivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocycles. Inparticular, proline analogues in which the ring size of the prolineresidue is changed from 5 members to 4, 6, or 7 members can be employed.Cyclic groups can be saturated or unsaturated, and if unsaturated, canbe aromatic or non-aromatic. Heterocyclic groups preferably contain oneor more nitrogen, oxygen, and/or sulfur heteroatoms. Examples of suchgroups include the furazanyl, furyl, imidazolidinyl, imidazolyl,imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl (e.g. morpholino),oxazolyl, piperazinyl (e.g., 1-piperazinyl), piperidyl (e.g.,1-piperidyl, piperidino), pyranyl, pyrazinyl, pyrazolidinyl,pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl(e.g., 1-pyrrolidinyl), pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl,thienyl, thiomorpholinyl (e.g., thiomorpholino), and triazolyl groups.These heterocyclic groups can be substituted or unsubstituted. Where agroup is substituted, the substituent can be alkyl, alkoxy, halogen,oxygen, or substituted or unsubstituted phenyl.

One can also readily modify peptides by phosphorylation, and othermethods (e.g., as described in Hruby, et al. (1990) Biochem J.268:249-262).

Treatment

Aspects disclosure relate to methods for treating a subject havingcancer. In some embodiments, the method comprises administering to asubject having cancer a cyclic Psap peptide as described herein. In someembodiments, the cyclic peptide comprises the amino acid sequence DWLPK(SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicates D-aminoacid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower case dindicates D-amino acid). In some embodiments, the amino acid sequence ofthe cyclic peptide is DWLPK (SEQ II.) NO: 1), dWLPK (SEQ ID NO: 3, lowercase d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK (SEQ IDNO: 4, lower case d indicates D-amino acid). In some embodiments, thecyclic peptide comprises the amino acid sequence DWLPK (SEQ ID NO: 1).In some embodiments, the amino acid sequence of the cyclic peptide isDWLPK (SEQ ID NO: 1).

Other aspects of the disclosure relate to compositions and uses ofcompositions in the manufacture of a medicament for treating a subjecthaving cancer. In some embodiments, the composition comprises a cyclicPsap peptide as described herein. e.g., a cyclic peptide having theamino acid sequence DWLPK (SEQ ID NO: 1).

As used herein, “treat” or “treatment” of cancer includes, but is notlimited to, preventing, reducing, or halting the development of acancer, reducing or eliminating the symptoms of cancer, suppressing orinhibiting the growth of a cancer, preventing or reducing metastasisand/or invasion of an existing cancer, promoting or inducing regressionof the cancer, inhibiting or suppressing the proliferation of cancerouscells, reducing angiogenesis and/or increasing the amount of apoptoticcancer cells.

Yet other aspects of the disclosure relate to methods for treating asubject having an inflammatory disease or disorder. In some embodiments,the inflammatory disease or disorder is inflammatory bowel disease(IBD), e.g., Crohn's disease. In some embodiments, the method comprisesadministering to a subject having an inflammatory disease or disorder(e.g., Crohn's disease) a cyclic Psap peptide as described herein. Insome embodiments, the cyclic peptide comprises the amino acid sequenceDWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3, lower case d indicatesD-amino acid). DWGPK (SEQ ID NO: 2), or dWGPK (SEQ ID NO: 4, lower cased indicates D-amino acid). In some embodiments, the amino acid sequenceof the cyclic peptide is DWLPK (SEQ ID NO: 1), dWLPK (SEQ ID NO: 3,lower case d indicates D-amino acid), DWGPK (SEQ ID NO: 2), or dWGPK(SEQ ID NO: 4, lower case d indicates D-amino acid). In someembodiments, the cyclic peptide comprises the amino acid sequence DWLPK(SEQ ID NO: 1). In some embodiments, the amino acid sequence of thecyclic peptide is DWLPK (SEQ ID NO: 1).

Other aspects of the disclosure relate to compositions and uses ofcompositions in the manufacture of a medicament for treating a subjecthaving an inflammatory disease or disorder, e.g., Crohn's disease. Insome embodiments, the composition comprises a cyclic Psap peptide asdescribed herein, e.g., a cyclic peptide having the amino acid sequenceDWLPK (SEQ ID NO: 1).

As used herein, “treat” or “treatment” of an inflammatory disease ordisorder includes, but is not limited to, preventing, reducing orhalting the development of an inflammatory disease or disorder orreducing or eliminating the symptoms an inflammatory disease ordisorder.

An effective amount is a dosage of the cyclic Psap peptide sufficient toprovide a medically desirable result, such as treatment of cancer or aninflammatory disease or disorder. The effective amount will vary withthe particular disease or disorder being treated, the age and physicalcondition of the subject being treated, the severity of the condition,the duration of the treatment, the nature of any concurrent therapy, thespecific route of administration and the like factors within theknowledge and expertise of the health practitioner. For administrationto a subject such as a human, a dosage of from about 0.001, 0.01, 0.1,or 1 mg/kg up to 50, 100, 150, or 500 mg/kg or more can typically beemployed.

In some embodiments, the effective amount is a dosage of the cyclic Psappeptide that causes no toxicity to the subject. In some embodiments, theeffective amount is a dosage of the cyclic Psap peptide that causesreduced toxicity to the subject as compared to a linear Psap peptide. Asused herein, the term “no toxicity” or “reduced toxicity” indicates thata cyclic Psap peptide does not induce or decreases the incidence ordegree of one or more adverse response(s) in a subject or in a cell,tissue or organ of a subject to which it is administered. For example, acyclic Psap peptide described herein does not cause dysfunction of anorgan or a system of organs or cause cell death. For example, a cyclicPsap peptide is not nephrotoxic, not toxic to the spleen and/or nothepatotoxic. Methods for measuring toxicity are well known in the art(e.g., biopsy/histology of the liver, spleen, and/or kidney; alaninetransferase, alkaline phosphatase and bilirubin assays for livertoxicity; and creatinine levels for kidney toxicity).

Cyclic Psap peptides and compositions thereof can be formulated for avariety of modes of administration, including systemic, topical orlocalized administration. A variety of administration routes areavailable. The particular mode selected will depend upon the type ofcancer being treated and the dosage required for therapeutic efficacy.The methods of the disclosure, generally speaking, may be practicedusing any mode of administration that is medically acceptable, meaningany mode that produces effective levels of the active compounds withoutcausing clinically unacceptable adverse effects. Such modes ofadministration include, but are not limited to, oral, rectal, topical,nasal, intradermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Thepharmaceutical compositions described herein are also suitablyadministered by intratumoral, peritumoral, intralesional or perilesionalroutes, to exert local as well as systemic effects.

Techniques and formulations generally can be found in Remington: TheScience and Practice of Pharmacy, Pharmaceutical Press; 22nd edition andother similar references. When administered, a Psap peptide may beapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. Pharmaceutical compositionsand pharmaceutically-acceptable carriers are also described herein. Suchpreparations may routinely contain salt, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the disclosure. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts.

In some embodiments, treatment with a cyclic Psap peptide may becombined with another therapy, such as a chemotherapy agent, radiation,a cytostatic agent, an anti-VEGF agent, an anti-angiogenesis factor, ap53 reactivation agent and/or surgery for cancer or an anti-inflammatoryagent for an inflammatory disease or disorder.

In some embodiments, a cyclic Psap peptide (e.g., DWLPK (SEQ ID NO: 1)may be used for stimulating expression of Tsp-1 in a subject in needthereof. In some embodiments, a method comprises administering to asubject an effective amount a cyclic Psap peptide to stimulateexpression of Tsp-1.

Compositions and Pharmaceutically-Acceptable Carriers

Other aspects of the disclosure relate to compositions comprising acyclic Psap peptide as described herein. In some embodiments, thecomposition is a pharmaceutical composition. In some embodiments, thecomposition comprises a cyclic Psap peptide as described herein and apharmaceutically-acceptable carrier. In some embodiments, thecomposition is for use in treating cancer or an inflammatory disease ordisorder. In some embodiments, the composition is for use in stimulatingTsp-1 in a subject in need thereof. In some embodiments, the compositioncomprises an additional agent, e.g., a chemotherapy agent, a cytostaticagent, an anti-VEGF agent, an anti-angiogenesis factor, a p53reactivation agent and/or an anti-inflammatory agent.

The term “pharmaceutically-acceptable carrier” as used herein means oneor more compatible solid or liquid filler, diluents or encapsulatingsubstances which are suitable for administration into a subject, e.g., ahuman. A pharmaceutically acceptable carrier is “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the tissue of the patient (e.g., physiologicallycompatible, sterile, physiologic pH, etc.). The term “carrier” denotesan organic or inorganic ingredient, natural or synthetic, with which theactive ingredient is combined to facilitate the application. Thecomponents of the pharmaceutical compositions also are capable of beingco-mingled with the molecules of the present disclosure, and with eachother, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy. Some examplesof materials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, methylcellulose,ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin. HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. The term “unit dose” when used in reference to apharmaceutical composition of the present disclosure refers tophysically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

The formulation of the pharmaceutical composition may dependent upon theroute of administration. Injectable preparations suitable for parenteraladministration or intratumoral, peritumoral, intralesional orperilesional administration include, for example, sterile injectableaqueous or oleaginous suspensions and may be formulated according to theknown art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution, suspension or emulsion in a nontoxic parenterallyacceptable diluent or solvent, for example, as a solution in 1,3propanediol or 1,3 butanediol. Among the acceptable vehicles andsolvents that may be employed are water, Ringer's solution, U.S.P. andisotonic sodium chloride solution. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordi-glycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. The injectable formulations can besterilized, for example, by filtration through a bacterial-retainingfilter, or by incorporating sterilizing agents in the form of sterilesolid compositions which can be dissolved or dispersed in sterile wateror other sterile injectable medium prior to use.

For topical administration, the pharmaceutical composition can beformulated into ointments, salves, gels, or creams, as is generallyknown in the art. Topical administration can utilize transdermaldelivery systems well known in the art. An example is a dermal patch.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the anti-inflammatory agent. Other compositionsinclude suspensions in aqueous liquids or non-aqueous liquids such as asyrup, elixir or an emulsion.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the anti-inflammatory agent, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and polyanhydrides. Microcapsules of theforegoing polymers containing drugs are described in, for example, U.S.Pat. No. 5,075,109. Delivery systems also include non-polymer systemsthat are: lipids including sterols such as cholesterol, cholesterolesters and fatty acids or neutral fats such as mono- di- andtri-glycerides; hydrogel release systems; sylastic systems; peptidebased systems; wax coatings; compressed tablets using conventionalbinders and excipients; partially fused implants; and the like. Specificexamples include, but are not limited to: (a) erosional systems in whichthe anti-inflammatory agent is contained in a form within a matrix suchas those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660 and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardwaredelivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions. Long-term release, areused herein, means that the implant is constructed and arranged todelivery therapeutic levels of the active ingredient for at least 30days, and preferably 60 days. Long-term sustained release implants arewell-known to those of ordinary skill in the art and include some of therelease systems described above.

In some embodiments, the pharmaceutical compositions used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). Alternatively, preservatives can be used toprevent the growth or action of microorganisms. Various preservativesare well known and include, for example, phenol and ascorbic acid. Thecyclic Psap peptide and/or the pharmaceutical composition ordinarilywill be stored in lyophilized form or as an aqueous solution if it ishighly stable to thermal and oxidative denaturation. The pH of thepreparations typically will be about from 6 to 8, although higher orlower pH values can also be appropriate in certain instances.

Subjects

Aspects of the disclosure relate to subjects, such as human subjects,having cancer and methods of treating such subjects. The cancer can bebenign or malignant, and it may or may not have metastasized. Any typeof cancer is contemplated herein, including, but not limited to,leukemias, lymphomas, myelomas, carcinomas, metastatic carcinomas,sarcomas, adenomas, nervous system cancers and genitourinary cancers.Exemplary cancer types include, but are not limited to, adult andpediatric acute lymphoblastic leukemia, acute myeloid leukemia,adrenocortical carcinoma, AIDS-related cancers, anal cancer, cancer ofthe appendix, astrocytoma, basal cell carcinoma, bile duct cancer,bladder cancer, bone cancer, osteosarcoma, fibrous histiocytoma, braincancer, brain stem glioma, cerebellar astrocytoma, malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, hypothalamic glioma, breast cancer, male breast cancer,bronchial adenomas, Burkitt lymphoma, carcinoid tumor, carcinoma ofunknown origin, central nervous system lymphoma, cerebellar astrocytoma,malignant glioma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colorectal cancer, cutaneous T-celllymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewingfamily tumors, extracranial germ cell tumor, extragonadal germ celltumor, extrahepatic bile duct cancer, intraocular melanoma,retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinalstromal tumor, extracranial germ cell tumor, extragonadal germ celltumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma,hairy cell leukemia, head and neck cancer, hepatocellular cancer,Hodgkin lymphoma, non-Hodgkin lymphoma, hypopharyngeal cancer,hypothalamic and visual pathway glioma, intraocular melanoma, islet celltumors. Kaposi sarcoma, kidney cancer, renal cell cancer, laryngealcancer, lip and oral cavity cancer, small cell lung cancer, non-smallcell lung cancer, primary central nervous system lymphoma, Waldenstrommacroglobulinema, malignant fibrous histiocytoma, medulloblastoma,melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous neckcancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosisfungoides, myelodysplastic syndromes, myeloproliferative disorders,chronic myeloproliferative disorders, nasal cavity and paranasal sinuscancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer,ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer,pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorialprimitive neuroectodermal tumors, pituitary cancer, plasma cellneoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer,rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterinesarcoma, Sezary syndrome, non-melanoma skin cancer, small intestinecancer, squamous cell carcinoma, squamous neck cancer, supratentorialprimitive neuroectodermal tumors, testicular cancer, throat cancer,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer,trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma,vaginal cancer, vulvar cancer, or Wilms tumor. In some embodiments, thecancer is melanoma or ovarian cancer.

Subjects having cancer may be identified using any method known in theart (e.g., blood tests, histology, CT scan, X-ray. MRI, physical exam,cytogenitic analysis, urinalysis, or genetic testing). A subjectsuspected of having cancer might show one or more symptoms of thedisease. Signs and symptoms for cancer are well known to those ofordinary skill in the art. Some exemplary laboratory tests include, butare not limited to, testing for cancer biomarkers such as cancer antigen(CA) 15-3, carcinoembryonic antigen (CEA) and HER-2 for breast cancer,human papillomavirus (HPV) E6 and E7 oncoproteins for cervical cancer,alpha-fetoprotein (AFP), AFP fractions L3, P4/5, and the +11 band, andultrasonography for hepatocellular carcinoma (HCC), prostate-specificantigen (PSA) for prostate cancer, and serum CA-125 for ovarian and HCC.

Other aspects of the disclosure relate to subjects, such as humansubjects, having inflammatory diseases or disorders and methods oftreating such subjects. Exemplary inflammatory diseases or disordersinclude, but are not limited to, rheumatoid arthritis, maculardegeneration (e.g., age-related macular degeneration, AMD), inflammatorybowel disease (IBD, e.g., Crohn's disease or ulcerative colitis),psoriasis, atherosclerosis, systemic lupus erythematosus, alopeciaareata, anklosing spondylitis, antiphospholipid syndrome, autoimmuneAddison's disease, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune inner ear disease, autoimmune lymphoproliferative syndrome(ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease,bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronicfatigue syndrome immune deficiency syndrome (CFIDS), chronicinflammatory demyelinating polyneuropathy, cicatricial pemphigoid, coldagglutinin disease, Crest syndrome, Dego's disease, dermatomyasitis,dermatomyositis—juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, grave's disease,guillain-barre, hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), Iga nephropathy, insulindependent diabetes (Type 1), juvenile arthritis, Meniere's disease,mixed connective tissue disease, multiple sclerosis, myasthenia gravis,pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychondritis, polyglancular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, Raynaud's phenomenon, Reiter's syndrome, rheumaticfever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome,Takayasu arteritis, temporal arteritis/giant cell arteritis, uveitis,vasculitis, vitiligo, and Wegener's granulomatosis. In some embodiments,the inflammatory disease or disorder is rheumatoid arthritis, maculardegeneration (e.g., age-related macular degeneration, AMD), inflammatorybowel disease (IBD, e.g., Crohn's disease or ulcerative colitis),psoriasis, or atherosclerosis.

Subjects having an inflammatory disease or disorder may be identifiedusing any method known in the art (e.g., blood tests, physical exam, CTscan, or MRI). A subject suspected of having an inflammatory disease ordisorder might show one or more symptoms of the disease or disorder.Signs and symptoms for inflammatory diseases or disorders are well knownto those of ordinary skill in the art.

In some embodiments, the inflammatory disease or disorder isinflammatory bowel disease (IBD), e.g., Crohn's disease. Subjects havingIBD, e.g., Crohn's disease, may be identified using any method known inthe art (e.g., blood tests, physical exam, fecal occult blood test,colonoscopy, flexible sigmoidoscopy, CT scan, MRI, capsule endoscopy,double balloon endoscopy, small bowel imaging, or a barium enema). Asubject suspected of having IBD might show one or more symptoms of thedisease. Signs and symptoms for IBD are well known to those of ordinaryskill in the art.

EXAMPLES Example 1

Cyclic versions of the peptides DWLPK (SEQ ID NO; 1), dWLPK (SEQ ID NO:3), DWGPK (SEQ ID NO: 2), and dWGPK (SEQ ID NO: 4) were synthesizedaccording to methods known in the art (lower case d indicates a D-aminoacid) and compared to a linear peptide dWIP (SEQ ID NO: 5, lower case dand l indicate D-amino acids).

Cells were cultured and contacted with 4 micrograms/mL or 10micrograms/mL of each peptide and the level of thrombospondin-1 (Tsp-1)activation was measured by Western blot. Tsp-1 has been previously shownto be activated by Saposin A protein. FIG. 2 shows that the cyclicpeptide DWLPK (SEQ ID NO: 1) was the most effective at increasing thelevel of Tsp-1, with dWGPK (SEQ ID NO: 4) being the next most effectiveand dWLPK (SEQ ID NO: 3) and DWGPK (SEQ ID NO: 2) being the leasteffective. This was surprising, as a similar study with linear versionsof these peptides revealed that linear DWGPK (SEQ ID NO: 2) was moreeffective than linear DWLPK (SEQ ID NO: 1).

The cyclic peptide DWLPK (SEQ ID NO: 1) was also tested for stability inhuman plasma compared to the linear peptide dWIP (SEQ ID NO: 5). Eachpeptide was incubated in human plasma at 37 degrees Celsius, for 2, 4,8, or 24 hours. It was found that the cyclic peptide retained theability to activate Tsp-1, even after 24 hours of incubation in theplasma (FIG. 3 ). The linear peptide had significantly less activity at24 hours, despite being stabilized against degradation by D-amino acidsubstitutions.

The cyclic peptide dWGPK (SEQ ID NO: 4) was then tested in a mouse modelof melanoma. For the melanoma model, 5×10⁵ B16-B16 melanoma cells wereinjected in syngeneic C57B16 mice. Mice were then treated with eitherthe cyclic peptide or linear dWIP (SEQ ID NO: 5) or control. In a firstexperiment, treatment with the peptides began about day 12 and thevolume of the tumor was measured over time up until about 20-25 dayspost cell injection (FIG. 4 ). In a second experiment, treatment withthe peptides began on day 15 and the volume of the tumor was measuredover time up until about 27 days post cell injection (FIG. 5 ). It wasfound that the cyclic peptide was four times as effective as the linearpeptide, as a dose of 10 mg/kg of the cyclic peptide was as effective asa dose of 40 mg/kg of the linear peptide (FIG. 4 ). This was confirmedin the second experiment (FIG. 5 ).

Next the cyclic peptide DWLPK (SEQ ID NO: 1) was tested in a mouse modelof ovarian cancer. For the ovarian cancer mouse model, 1 millionpatient-derived ovarian cancer cells expressing luciferase were injectedintraperitoneally into mice. The tumor burden was measured in vivo usingluciferase. Treatment with the cyclic peptide began after 40 days andwas administered for 10 days at 10 mg/kg. After 10 days of treatment,the mice were euthanized and the tumor was analyzed histologically. Asshown in FIG. 6 , the luciferase output in mice treated with the cyclicpeptide went down over the 10 days of treatment, indicated regression ofthe tumor. All cyclic peptide treated tumors expressed Tsp-1 and were<25% the size of untreated tumors upon observation by histology.

It was also shown that the cyclic peptide DWLPK (SEQ ID NO: 1)stimulated Tsp-1 in macrophages in mice (FIG. 7 ), indicating that thecyclic peptide could also induce expression of Tsp-1 in the stroma.

Lastly, it was shown that dosage with the cyclic peptide DWLPK (SEQ IDNO: 1) did not cause any toxicity to the liver or spleen, as measuredusing histology (FIG. 8 ).

In summary, these data show that cyclic Psap peptides were more stableand more effective than linear Psap peptides. In addition, the cyclicpeptide DWLPK (SEQ ID NO: 1) appeared to have the best activity in vitroand did not induce toxicity.

Example 2

A model of Crohn's disease was used to test the efficacy of the cyclicpeptide DWLPK (SEQ ID NO: 1) for treating Crohn's disease. The cyclicpeptide was tested in a Dextran Sodium Sulfate (DSS)-induced model ofCrohn's Disease where DSS was given to the mice in their drinking water(3.5% weight:volume) for 7 days. In one group the mice were treatedconcurrently with the cyclic peptide and DSS, while the other group wastreated with DSS alone. The cyclic peptide significantly reducedinflammation in this model (FIG. 9 ). The mice treated with cyclicpeptide also lost less weight than the mice treated with only DSS (FIG.10 ). Lastly, the cyclic peptide was shown to stimulate Tsp-1 andinhibit macrophage infiltration in these mice (FIG. 11 ). These datasuggest that cyclic Psap peptides, in particular cyclic DWLPK (SEQ IDNO: 1), are effective for treating Crohn's disease.

Example 3

(i) A model of AMD is used to test the efficacy of a cyclic Psap peptidedescribed herein in treating AMD. Lesions are created on a mouse'sretina with a laser. The mice are then treated with cyclic Psap peptideas described herein, e.g., 10 or 40 mg/kg of cyclic DWLPK (SEQ ID NO:1), or a scrambled peptide control. Treatment is either systemically(e.g., by intravenous or intraperitoneal injection) or by intravitreousinjection. The rate of healing of the lesion is measured over time. Itis expected that the lesion will heal faster in mice treated with thecyclic Psap peptide than in mice treated with the control.

(ii) A rodent model of collagen induced arthritis (CIA), an autoimmunemodel that resembles rheumatoid arthritis, is used to test the efficacyof a cyclic Psap peptide described herein for treating rheumatoidarthritis. CIA is inducible in inbred DBA/1 male mice by primingintradermally with heterologous or homologous collagen U (about 50microgram) in Freunds complete adjuvant and 2 weeks later boosting withthe same amount of collagen II in Freunds incomplete adjuvant. Thearthritis develops approximately 3 weeks after the priming dose andreaches its maximum within 8 weeks post priming. The mice have highlevels of collagen H specific antibodies, collagen II specific T cellsas well as signs of systemic inflammation (e.g. production of IL-6, TNFetc.). Locally in the joints one observes both overwhelming inflammatoryinfiltrates (consisting of T cells, macrophages, neutrophils andfibroblasts) as well as severe destruction of cartilage and subchondralbone. These features mimic well the process seen in human rheumatoidarthritis (Myers et al., Life Sciences 61, p 1861-1878, 1997). The miceare then treated with cyclic Psap peptide as described herein, e.g., 10or 40 mg/kg of cyclic DWLPK (SEQ ID NO: 1), or a scrambled peptidecontrol. Treatment is either systemically (e.g., by intravenous orintraperitoneal injection) or by local administration. Severalparameters of the disease (e.g., clinical signs and symptoms, onset,progression, severity, and remission of symptoms) are measured. It isexpected that one or to more of these parameters will be improved (e.g.,decreased clinical signs and symptoms, delayed onset, slowedprogression, reduced severity, and/or remission) in mice treated withthe cyclic Psap peptide than in mice treated with the control.

(iii) A model of psoriasis is used to test the efficacy of a cyclic Psappeptide described herein in treating psoriasis. Imiquimod (IMQ) isapplied to the skin of mice to induce psoriasis-like dermatitis (see.e.g., van der Fits et al. Imiquimod-induced psoriasis-like skininflammation in mice is mediated via the IL-23/IL-17 axis. (2009) J.Immunol.; 182(9):5836-45). The mice are then treated with cyclic Psappeptide as described herein, e.g., 10 or 40 mg/kg of cyclic DWLPK (SEQID NO: 1), or a scrambled peptide control. Treatment is eithersystemically (e.g., by intravenous or intraperitoneal injection) ortopically. The rate of healing of the dermatitis is measured over time.Epidermal expression of IL-23, IL-17A, and IL-17F is also measured atthe conclusion of the experiment. It is expected that the dermatitiswill heal faster and the levels of IL-23, IL 17A, and/or IL 17F will bedecreased in mice treated with the cyclic Psap peptide compared to micetreated with the control.

(iv) A model of atherosclerosis is used to test the efficacy of a cyclicPsap peptide described herein in treating atherosclerosis. Mice lackingthe apoE or the LDL receptor (LDLR) gene are used as a model foratherosclerosis. The mice are fed a high-fat, high-cholesterol Westerntype diet or regular chow for 8 weeks or more, preferably at least 15weeks. The mice are treated with cyclic Psap peptide as describedherein, e.g., 10 or 40 mg/kg of cyclic DWLPK (SEQ ID NO: 1), or ascrambled peptide control. Treatment is systemically delivered (e.g., byintravenous or intraperitoneal injection). Lesions that develop in theaortic root, the innominate artery (brachiocephalic) and other branchesof the aorta, as well as the pulmonary and carotid arteries are measuredin the mice after at least 8 weeks. It is expected that the lesions willheal faster in mice treated with the cyclic Psap peptide than in micetreated with the control.

Example 4

Abstract

Virtually 100% of ovarian cancer-related deaths are caused by metastaticdissemination of cells from the primary tumor resulting in subsequentorgan failure. However, despite the increased understanding into thephysiological processes involved in tumor metastasis, there are noclinically approved drugs that have shown significant efficacy attreating advanced, metastatic, ovarian cancer. Psap has been identifiedas a potent inhibitor of tumor metastasis via stimulation of p53 and theanti-tumorigenic protein thrombospondin-1 (Tsp-1) in bone marrow derivedcells that are recruited to metastatic sites. It is demonstrated that˜100% of human serous ovarian tumors express C036, the receptor thatmediates the pro-apoptotic activity of Tsp-1. A peptide derived fromPsap which would be effective in treating this form of ovarian cancerwas investigated. The activity and stability of the peptide wasinvestigated by developing a novel cyclic peptide with drug-likeproperties derived from Psap. The cyclic Psap peptide was able tosignificantly regress a PDX model of metastatic ovarian cancer.

Introduction

Ovarian cancer is the most lethal gynecologic malignancy and the fourthleading cause of cancer deaths in women. Pathologically, ovarian canceris categorized into multiple subtypes, with epithelial ovarian cancer(EOC) representing 90% of cases. Despite our increased understanding ofthe biology governing the progression of EOC the survival rate forpatients with stage IV EOC is only 17%. As such, there is a need forefficacious therapies that can specifically treat advanced, metastaticovarian cancer. While many patients display a response to platinumagents as first line therapies, 70% percent develop resistance (1, 2).Currently for these patients there are no approved therapies thatsignificantly increase overall survival.

It has been previously reported that the development of a small peptidederived from Psap potently inhibits tumor metastasis in multiple typesof tumor models (3, 4). Specifically, Psap, and the peptide derived fromit, inhibits tumor metastasis by stimulating the broadly actinganti-tumorigenic protein Thrombospondin-1 (Tsp-1) inCD11b⁺/Gr1⁺/Lys6C^(hi) monocytes (3). These monocytes are recruited tosites of future metastatic lesions, termed premetastatic niches, wherethey persist after colonization and stimulate tumor growth. However,systemic administration of the Psap peptide stimulates the production ofTsp-1 in these cells, which renders the sites refractory to futuremetastatic colonization (3). These results demonstrate that stimulationof Tsp-1 in the tumor microenvironment could prevent future metastaticevents. Many ovarian cancer patients present at first diagnosis withmetastatic disease. As such, a therapeutic agent that can regress, oreven stabilize, metastatic lesions is needed.

It is demonstrated that stimulating Tsp-1 in the microenvironment of ametastatic, platinum resistant, ovarian cancer PDX model can inducesignificant regression of established lesions. Such a striking effect isachieved due to the fact that serous ovarian cancer cells express thereceptor for Tsp-1, CD36, that mediates the proapoptotic effectpreviously observed in endothelial cells (5).

Results

Incorporation of d-Amino Acids Increases the Activity of a Psap PeptideIn Vivo

Identification of both a 4- and 5-amino acid peptide derived from thesaposin A domain of Psap that was able to inhibit both a tail vein modelof Lewis Lung Carcinoma metastasis and in an adjuvant model of breastcancer metastasis when administered systemically was previouslydescribed (3). The therapeutic efficacy of peptides is often limited bytheir stability, or resistance to degradation by proteases. One commonmethod of increasing the stability of peptides in vivo is to incorporated-amino acids into the sequence, since d-amino acids are notincorporated into naturally occurring proteins, proteases do notrecognize them as substrates (6-10). The stability of the 4-amino acidPsap peptide by incorporating d-amino acids at different moieties wasinvestigated. Two peptides with d-amino acids incorporated, incombination, at the first (aspartate) and third (leucine) or the second(tryptophan) and fourth (proline) residues were synthesized. Theactivity of these peptides along with the native 1-amino acid peptide invitro by measuring their ability to stimulate thrombospondin-1 (Tsp-1)in WI-38 lung fibroblasts was tested. By western blot analysis, it wasfound that there was no difference in the stimulation of Tsp-1 betweenthe three peptides in vitro (FIG. 12A).

The activity of the 1,3-d-amino acid Psap peptide and the native Psappeptide were tested in vivo. The peptides were systematicallyadministered to C57BL6/J mice that were pretreated with conditionedmedia (CM) from PC3M-LN4 (LN4) cells, which previously mimicked thesystemic properties of metastatic tumors by repressing the expression ofTsp-1 in the lungs of mice (3, 4). After 3 days of treatment with LN4 CMalone or in combination with two different doses (10 mg/kg/day and 30mg/kg/day) of d- and 1-amino acid peptides (independently) protein poolswere prepared from the harvested lungs of each treatment group. Thelevel of Tsp-1 expression in the lungs of these mice were assessed bywestern blot analysis. It was observed that the 1,3-d-amino acid peptidestimulated Tsp-1 3-fold greater than the native peptide (FIG. 12B). Adose of 10 mg/kg of the native peptide did not significantly stimulateTsp-1 (FIG. 12B). Conversely, a dose of 10 mg/kg of the d-amino acidpeptide stimulated Tsp-1 to the same degree as a dose of 30 mg/kg of thenative peptide and a dose of 30 mg/kg of the d-amino acid peptidestimulated Tsp-1 approximately 3-fold greater than the same dose ofnative peptide. Based on the observation that the in vitro activity ofthe two peptides was virtually identical, it was concluded that thedifference in activity in vivo was due to a difference in stability.

Human Serous Ovarian Cancer Cells are Sensitive to Tsp-1 MediatedKilling

In order to test the efficacy of the d-amino acid Psap peptide, asuitable tumor model that would represent a potential clinicalapplication for the peptide, was determined. Given that Psap, and thepeptide derived from it, stimulates Tsp-1 in bone marrow derived cellsthat are recruited to sites of metastasis, a specific type of cancerthat expressed the receptor for Tsp-1 that mediates its pro-apoptoticactivity, CD36 was sought to be identified (5). It has been reportedthat serous ovarian epithelial cells and human ovarian cancer cellsexpress CD36 (11-13). Fourteen primary human ovarian cancer cell linesderived from the ascites of patients with platinum resistant ovariancancer for expression of CD36 were surveyed. All of the patient derivedcells expressed levels of CD36 that were approximately equivalent to thelevel expressed by human microvascular endothelial cells (FIG. 12C).Three of these cell lines were treated with recombinant human Tsp-1 forup to 72 hours and determined its effect on cell number and apoptosis.RhTsp-1 treatment resulted in a decrease in total cell number for allthree cell lines of up to 50% of the original number of cells plated(FIG. 12D). Moreover, by FACS analysis 30-60% of treated cells wereobserved to be apoptotic following Tsp-1 treatment, as defined by AnexinV positivity (FIG. 12E). In contrast, it was observed that followingcisplatin treatment a much greater percentage of ovarian cancer cellsunderwent necrosis, as defined by low Anexin V and high propidium iodide(PI) staining (FIG. 12E). These findings suggest that ovarian cancercells may respond favorably to treatment with the Psap peptide.

The Psap Peptide Regresses an Established PDX Model of Ovarian CancerMetastases

It was previously demonstrated that Psap and the Psap peptide couldinhibit the formation of metastases (3, 4). 75 percent of ovarian cancerpatients already have disseminated disease at the time of firstdiagnosis (E. Lengyel, Ovarian cancer development and metastasis. Am JPathol 177, 1053-1064 (2010)). For these patients inhibiting metastasiswould have limited therapeutic benefit. Rather, these patients require atherapeutic agent that can regress or, at the very least, stabilizeexisting metastases. Whether the d-amino acid Psap peptide could havetherapeutic efficacy in a model of established metastatic disseminationwas investigated. 1×10⁶ DF14 cells, expressing firefly luciferase, wereinjected into the peritoneal cavity of SCID mice to mimic the route ofdissemination of human ovarian cancer. The growth of metastatic coloniesin the mice in real time were monitored via relative luciferaseintensity and when the average intensity of the luciferase signal was0.5-1×10⁸ RLU, treatment with vehicle (saline), the d-amino acid peptide(40 mg/kg QD), or cisplatin (4 mg/kg QOD) was started. Both the peptideand cisplatin were able to regress tumor volume, as determined byluciferase intensity, for the first 20 days of treatment (FIGS. 13A and13B). However, during those 20 days half of the cisplatin treated micedied from adverse side effects of the drug as defined by total bodyweight, which decreased by 40% (FIG. 13C). Moreover, after 20 days thetumors in the group of cisplatin treated mice that survived began togrow, despite continued treatment with cisplatin, and all the remainingmice died within 10 days (FIG. 13A).

Conversely, no loss of body weight was observed in the group of peptidetreated mice (FIG. 13C) and the tumors continued to shrink until by day48 there was no detectable luciferase signal in any of the mice (FIGS.13A and 13B and FIG. 16 ). These mice were treated for an additional 35days (83 days in total) until the control treated group displayedconditions associated with morbidity. During this treatment time theluciferase signal never re-emerged and gross examination of the micerevealed no metastatic lesions (FIGS. 13A and 13D). The livers andspleens of the Psap peptide treated mice were examined histologically(H&E) to determine whether there were any micrometastases. Metastaticlesions in the Psap peptide treated mice were not identifiable (FIG.13E).

Metastases in the peritoneal cavity recruited Cd11b⁺/Gr1⁺ bone marrowderived cells, analogous to lung metastases was investigated (3).Ascites fluid were collected from the peritoneal cavity of controltreated mice bearing DF14 metastases and fluid from the peritonealcavity of Psap peptide treated mice that showed no signs of metastases.The fluid was FACS sorted from these mice to assess the population ofCd11⁺/Gr1⁺ bone marrow derived cells. The FACS analysis revealed that71-77% of the cells in the peritoneal fluid of control treated mice wereCd11b⁺/Gr1⁺ (FIG. 13E and FIG. 16 ) while only 31.4% of the cells in theperitoneal fluid from peptide treated mice were Cd11b⁺/Gr1⁺ (FIG. 13E).Based on these findings it was concluded that the Psap peptide was ableto dramatically regress established metastases to the point where nodetectable lesions could be found.

Cyclization Further Stabilizes the Psap Peptide while Increasing itsActivity

While the results of the peptide treatment of the PDX model of ovariancancer were very promising it was postulated that the stability andactivity of the peptide could be increased even more. The peptidederived from Psap was located in a region of the protein that containeda 13-amino acid loop between two helices that was stabilized by adisulfide bond (3). As such a 5-amino acid peptide that was cyclized viabackbone (N—C) cyclization was synthesized (FIG. 14A, cyclic DWLPK (SEQID NO: 1)). The activity of this peptide in vitro was tested based onits ability to stimulate Tsp-1. The cyclic peptide stimulated Tsp-13-fold greater than the d-amino acid linear peptide (FIG. 14B).

Cyclization also has the effect of increasing the stability of peptidesby forcing them into a conformation that is not recognized by mostnaturally occurring proteases (9, 10, 14-18). The stability of thecyclic peptide to the linear d-amino acid peptide in human plasma wascompared. Both peptides in human plasma were incubated at 37° C. for upto 24 hours and then the ability of the plasma/peptide mixture tostimulate Tsp-1 in WI-38 fibroblasts was tested. The level of secretedTsp-1 was measured following treatment with the peptide/plasma mixtureby ELISA and found that the stimulation of Tsp-1 by the two peptides wasroughly equivalent after up to 8 hours of incubation (FIG. 14C).However, after 24 hours of incubation in human plasma, the cyclicpeptide retained greater than 70% of its Tsp-1 stimulating activity,while the plasma containing linear peptide was no longer able tostimulate Tsp-1 (FIG. 14C). As such, it was concluded that the cyclicpeptide was significantly more active and stable than the linear d-aminoacid peptide.

Based on these findings the efficacy of the cyclic peptide using theDF14 model was tested. In order to better study the effects of thepeptide on the metastatic lesions mice were injected with 1×10⁷ cellsand allowed the luciferase signal to reach 9×10⁹ RLU. The mice weretreated with the cyclic peptide for only 10 days to ensure that therewould be sufficient tumor tissue to analyze. Significantly, after only10 days of treatment, the average luciferase signal in the peptidetreated mice decreased to 4×10⁸ (FIG. 14D). The liver, spleen andomentum were analyzed of both the peptide and control treated mice byH&E, and IHC for Tsp-1, Gil and TUNEL. The metastatic lesions in thecontrol treated mice were, on average, 2.3-fold larger than those in thepeptide treated mice (FIG. 14E). Consistent with the mechanism of action(MOA) of the Psap peptide, all of the peptide treated tumors stainedpositive for Tsp-1 (FIG. 14F) expression. Also consistent with previousobservations, it was found that all of the Tsp-1 expressing cells werealso Gr1 positive (FIG. 14G) (3). Moreover, all of the lesions in thepeptide treated mice contained a significant percentage of TUNELpositive cells, with an average of 40% TUNEL positive cells/lesion(FIGS. 14H and 14I). Conversely, control treated tumors contained, onaverage, only 1.4% TUNEL positive cells/lesion (FIGS. 14H and 14I).

Metastatic Serous Ovarian Tumors Express Lower Levels of Psap but HigherCD36 Expression than Primary Tumors

It was previously demonstrated that the activity of the Psap peptidesagainst tumors formed by patient derived ovarian cancer cells expressedCD36. The prevalence of CD36 expression in human ovarian cancerpatients, and thus how widely applicable a potential Psap-basedtherapeutic agent would be for this disease was investigated. It waspostulated that prosaposin expression should decrease as tumors progressto the metastatic stage. Accordingly, a tumor tissue microarray (TMA)comprised of 139 patients with metastatic serous ovarian cancer andnormal ovarian tissue from 46 patients was utilized. The tissue for CD36and Psap expression was stained and then scored the intensity using thestaining index (SI) method (R. Catena et al., Bone marrow-derived Gr1+cells can generate a metastasis-resistant microenvironment via inducedsecretion of thrombospondin-1. Cancer Discov 3, 578-589 (2013)). 61% oftissue from normal ovaries expressed CD36 with an average SI of 2.39(out of a possible maximum score of 9) (FIGS. 15A-15C, 15M and Table 1).Analysis of 134 primary ovarian tumors revealed that 97% (130/134) oftumors stained positive for CD36 with an average SI of 5.32 (FIGS.15D-15F, 15M and Table 1). 121 visceral metastases from the 134 patientswere examined. 97% (117/121) of the metastatic lesions stained positivefor CD36 with an average SI of 6.61 (FIGS. 15G-15I, 15M and Table 1).Finally, 100% of lymph node metastases (13/13) stained positive for CD36with an average SI of 6.69 (FIGS. 15J-15L, 15M and Table 1).

TABLE 1 CD36 expression in human ovarian cancer patient TMA StainingIndex % Positive Samples % Samples > 6 Normal 2.39 62 28.3 PrimarySerous EOC 5.38 94 79.9 Visceral Metastases 6.61 97 91.7 Lymph node 6.6997 92.3 metastases

Expression of prosaposin in human ovarian cancer patients wasinvestigated with the speculation that levels should decrease with tumorprogression based on its mechanism of action. Prosaposin levels in theovarian cancer TMA samples were examined. Normal ovaries expressedrelatively low levels of prosaposin with an average SI of 4.29 (FIG. 15Nand Table 2). Primary ovarian tumors expressed higher levels ofprosaposin, with an average SI of 5.17 (FIG. 15N and Table 2).Strikingly, in visceral and lymph node metastases prosaposin levelsdropped significantly, with average SI's of 4.14 and 4.07 respectively(FIG. 15N and Table 2), Thus, when taken together, CD36 expression isincreased in primary ovarian tumors compared to normal ovaries and thatCD36 expression in metastatic lesions is further increased compared toprimary tumors, while prosaposin expression decreases with tumorprogression. These findings suggest that metastatic ovarian tumorsrepress prosaposin expression but retain CD36, which could be used toeffectively target ovarian cancer with a prosaposin-derived therapy.

TABLE 2 Prosaposin expression in human ovarian cancer patient TMA PsapStaining Index P-value Normal 4.3 N/A Primary Serous EOC 5.17 N/AVisceral Metastases 4.14 <0.001 Lymph node metastases 4.07 0.017Discussion

It was previously demonstrated that prosaposin and a short 5-amino acidpeptide derived from it can potently inhibit metastasis (3, 4). Thedevelopment process of a cyclic peptide derived from prosaposin isdelineated with significantly greater activity and stability than thenative peptide. It was demonstrated that incorporation of d-amino acidsat the first and third residues of the native linear peptide increasesin vivo activity. The peptide was further modified to make it moredrug-like by cyclizing a 5-amino acid peptide via backbone N—Ccyclization. The cyclic peptide displays even greater in vivo activitythan the d-amino acid linear peptide It was demonstrated that bothmodified peptides can potently regress established metastases in a PDXmodel of ovarian cancer. The cells used in this PDX model were derivedfrom platinum resistant patients, the most common first line treatmentfor ovarian cancer patients.

Through an analysis of human ovarian cancer cell lines derived directlyfrom patient ascites it was found that all tested cell lines expressCD36, the receptor for Tsp-1, the downstream target of Psap and thepeptide in bone marrow derived cells. It was also demonstrated thatrecombinant Tsp-1 induces apoptosis in these CD36 expressing serousovarian cancer cells. The in vitro activity of Tsp-1 on ovarian cancercells was recapitulated in a PDX model of ovarian cancer in which thecyclic Psap peptide stimulated the expression of Tsp-1 in Cd11b⁺/Gr1⁺bone marrow derived cells, which were recruited to the peritoneal cavityof metastasis bearing mice. The induced expression of Tsp-1 in thesecells resulted in a significant induction of apoptosis in the tumorcells and significant regression.

Based on these results it is believed that the Psap peptide has thepotential to inhibit ovarian cancer progression via three distinctmechanisms, all mediated by the induction of Tsp-1. The first,demonstrated here, is the direct cell killing mediated by downstreamsignaling from CD36 triggered by Tsp-1. The second is via the widelyestablished anti-angiogenic activity of Tsp-1 (D. J. Good et al., Atumor suppressor-dependent inhibitor of angiogenesis is immunologicallyand functionally indistinguishable from a fragment of thrombospondin.Proc Natl Acad Sci USA 87, 6624-6628 (1990)). The third is via Tsp-1binding to its other cell surface receptor. CD47, and blocking the “donot eat me” signal mediated by CD47 binding to SIRPα on macrophages (P.Burger, P. Hilarius-Stokman, D. de Korte, T. K. van den Berg, R. vanBruggen, CD47 functions as a molecular switch for erythrocytephagocytosis. Blood 119, 5512-5521 (2012); A. Saumet, M. B. Slimane, M.Lanotte, J. Lawler, V. Dubernard, Type 3 repeat/C-terminal domain ofthrombospondin-1 triggers caspase-independent cell death throughCD47/alphavbeta3 in promyelocytic leukemia NB4 cells. Blood 106, 658-667(2005)).

Finally, an analysis of tumor tissue from 134 patients with serousovarian cancer revealed that 97% expressed CD36 and that this expressionwas not only maintained in metastatic lesions, but that the level ofCD36 expression actually increased with tumor progression. The mostcommon first line therapeutic strategy for ovarian cancer patients isplatinum-based chemotherapy (E. Lengyel. Ovarian cancer development andmetastasis. Am J Pathol 177, 1053-1064 (2010)). 70% of ovarian cancerpatients develop resistance to this treatment (E. Lengyel, Ovariancancer development and metastasis. Am J Pathol 177, 1053-1064 (2010)).For these patients there is no effective FDA approved therapeutic agentand, as such, the survival rate is ˜17% (E. Lengyel, Ovarian cancerdevelopment and metastasis. Am. J Pathol 177, 1053-1064 (2010)). Thefindings presented here suggest that a Psap-based therapeutic agentcould have significant efficacy for the vast majority of ovarian cancerpatients based on its mechanism of action and the pervasiveness of CD36expression in the tumor cells of these patients.

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Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present disclosure toits fullest extent. The specific embodiments are, therefore, to beconstrued as merely illustrative, and not limitative of the remainder ofthe disclosure in any way whatsoever. All publications cited herein areincorporated by reference for the purposes or subject matter referencedherein.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

From the above description, one skilled in the all can easily ascertainthe essential characteristics of the present disclosure, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the disclosure to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A method for reducing inflammation in a subjectin need thereof, the method comprising: administering to the subject aneffective amount of a cyclic peptide, wherein the amino acid sequence ofthe cyclic peptide is selected from the group consisting of DWGPK (SEQID NO: 2) and dWGPK (SEQ ID NO: 4).
 2. The method of claim 1, whereinthe inflammation is associated with a disease or disorder selected fromthe group consisting of rheumatoid arthritis, age-related maculardegeneration (AMD), Crohn's disease, psoriasis, and atherosclerosis. 3.The method of claim 2, wherein the disease or disorder is Crohn'sdisease.
 4. The method of claim 1, wherein the peptide is linked to apolymer that enhances the serum half-life.
 5. The method of claim 4,wherein the polymer is selected from the group consisting of polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleicanhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran sulfate,polypropylene glycol, polyoxyethylated polyol, heparin, heparinfragments, polysaccharides, cellulose, methylcellulose, carboxymethylcellulose, starch, polyalkylene glycol and derivatives thereof,copolymers of polyalkylene glycols and derivatives thereof, polyvinylethyl ethers, and α,β-Poly(2-hydroxyethyl)-DL-aspartamide.